Japan is becoming an aging society with the highest average life expectancy in the world. People are beginning to place more emphasis on living better, i.e., “quality of life” (QOL), rather than merely prolonging life. One area that is continuing to attract attention is motor dysfunction. Arthritis includes a wide variety of diseases that lead to motor dysfunction; and in the United States, more than 70 million patients visiting hospitals in 2002 complained of having symptoms of some type of arthritis or chronic arthropathia. Currently, one out every three adults has this disease. Moreover, this number is predicted to double by the year of 2020. As a medical problem it is second only to heart disease, and costs 86.2 billion dollars in medical expenses per year. Among visiting patients, more than 20 million people over the age of 45 have osteoarthritis, which is one of the most common forms of arthritis. In Japan, a large number of people are suffering from the onset of osteoarthritis, with a prevalence rate of 30% for people of age 45 to 65, and 63% to 85% for people over the age of 65. One million patients in Japan are suffering from osteoarthritis, and the number of new patients is expected to increase by 900,000 every year. Motor system diseases such as osteoarthritis, and the like, differ from diseases of organs, in that they are rarely life threatening. However, these diseases may limit movement in the limbs, and thereby remarkably decrease a person's QOL (quality of life). Such motor system diseases are predicted to increase dramatically in the future, due to an increasingly aging population, and personal and social problems resulting from these disorders will continue to be extremely important issues.
The majority of these types of motor system diseases are the result of inflammation or an injury to cartilage tissue or osseous tissue. Currently, in cases of a severe disease, artificial joints containing metals and a ultrahigh molecular weight polyethylene are employed as a treatment. However, an artificial joint wears out within about ten years after implantation; and various undesirable biological reactions can be caused by abrasive powders. Although, research is being conducted on improving abrasive resistance in order to solve these problems, limitations in abrasive resistance are expected. Also, treatments of cartilage or osseous tissue in which tissue regenerative engineering technique are utilized as new solutions are drawing attention. Such a treatment method includes a method in which cultured chondrocytes or osteocytes, and cartilage or osseous tissue produced thereof, are transplanted to the affected area of the patient.
In 1994, Brittberg, et al., reported a treatment method, whereby articular cartilage tissue was extracted from an unloaded portion of a joint, and isolated cartilage tissue cells were cultured and transplanted to full-thickness defect of the damaged cartilage (Brittberg, et al., New England Journal of Medicine, 331(14), 889 (1994)). Since approval of this treatment method by the FDA in 1997, it has been commercialized and performed in more than 20,000 cases around the world. A study of 219 cases over a period of 2 to 10 years confirm that performance of the treatment method from medium- and long-term standpoint was excellent, showing functional improvement in 89% of the cases (Peterson L., 6th Annual Meeting, American Academic Orthopedic Surgery (1998)). On the other hand, in 2002, cases of fatality due to bacterial infection after transplantation were reported, and a CDC investigation discovered 41 cases of postoperative infection, in Japan also. Information regarding such cases was provided by the Ministry of Health, Labor and Welfare, Health Service Bureau, to the Japanese Orthopaedic Association, which stated that such a problem had to be reaffirmed, and caution adopted in processing. Moreover, this method cannot be utilized in the treatment of osteoarthritis accompanying a broad range of partially defective and degenerative cartilage or osseous tissues, and therefore, improvement is needed.
In Japan also, cartilage tissue is reconstructed by tissue-engineering using isolated chondrocytes from articular cartilage of an unloaded portion or bone marrow-derived mesenchymal stem cells, and clinical applications directed to osteochondral full-thickness defects have begun. However, examples of the abovementioned clinical applications are related to traumatic osteochondral injuries and osteochondral dissecans, and this treatment is limitedly applied only to cases where a very small area of cartilage defect is present (Japanese Patent Application No. 2001-384446, Japanese Patent Application No. 2002-216561, Japanese Patent Application No. 2003-358118). Currently, since treatment performance in artificial joint replacement technique is stable, it would appear that no progress is being made in the treatment of osteoarthritis accompanying a broad range of degeneration or defect of cartilage or osseous tissues. Furthermore, since these techniques require a scaffold made of a protein, sugar, or artificial polymer, etc., other than one produced from cultured cells, the biological effects of these scaffolds are also raising new problems. Therefore, there is a definite need for the development of a technique which does not employ this type of scaffold.
On the other hand, Hunziker et al. intensively addressed fundamental research into a treatment for osteoarthritis, by focusing on the pathology of osteoarthritis in defects that do not extend to cartilage degeneration and subchondral bones, and conducting fundamental research employing a model for partial articular cartilage defects. In their research, they discovered that cartilage repair and regeneration was the central role of synovium cells, and not chondrocytes (Hunziker et al., The Journal of Bone and Joint Surgery, 78-A, 721 (1996)). However, since this technique is not necessarily practical, and has a limited range for potential treatments, the discussion in the Hunziker et al. does not directly provide for a treatment of oseteoarthritis. Therefore, there is a strong need for early establishment of this technique for treatment of osteochondral defects.
Conventionally, cell culture is conducted on a glass surface, or on the surface of a synthetic polymer compound along with a variety of surface processing. In order to achieve this, for example, various types of vessels made of polystyrene subjected to surface processing such as silicone coating, gamma irradiation, etc., are commonly used as vessels for cell culture. Cells that have been cultured and grown with these types of cell culture vessels, are detached and harvested from the surface of the vessel by a chemical agent treatment or a proteinase treatment such as trypsin. However, in cases where the cells are harvested by the abovementioned chemical agent treatment, some disadvantages have been pointed out: the treatment method is cumbersome and complicated; the potential for contamination by impurities is increasing; and examples of defects, in which cells are caused to degenerate or are damaged by the chemical treatment, and lose their original function.
Thus far, in order to overcome the abovementioned disadvantages, a number of techniques have been proposed by the present inventors. Especially, in Japanese Patent Application No. 2001-226141, a method for producing a cultured cell sheet which comprises steps of coating the surface of the cell cultureware with a temperature responsive polymer having a lower or upper critical solution temperature ranging from 0° C. to 80° C. in water, having the cultured cell layers multi-layered by way of a conventional method, as necessary, and detaching the cultured cell sheet only by changing the temperature of the cultureware. As a result of application of this method, a cultured cell sheet having sufficient strength without a scaffold, other than that produced from the cultured cells, can be produced. Furthermore, a thus obtained cultured cell sheet also retains basal membrane-like proteins, and also has improved adhesiveness to tissue, when compared with a cell sheet harvested using the above described dispase treatment. Moreover, PCT International Publication No. WO 02/08387 discloses a method for producing a cultured myocardial cell sheet, which comprises steps of culturing the cells of myocardial tissue on a cell cultureware having a cultureware surface coated or covered with a temperature responsive polymer, preparing a myocardium-like cell sheet, and subsequently, adjusting a temperature of the culture medium to a temperature greater than the upper critical solution temperature or less than the lower critical solution temperature, bringing the layered cultured cell sheet into close contact with a polymer membrane, detaching the cultured intact cell sheet together with the polymer membrane, and three-dimensionally structuring by a predetermined method. As a result of application of this method, a myocardium-like cell sheet and a three-dimensional structure were discovered to be constructed in vitro with reduced structural defects and with some of the functions of myocardial tissue.
However, none of these methods has been investigated with regard to application in technology aimed at regeneration therapy for cartilage and osseous tissue.